本文選題：海兔素 + 酒精性肝病��； 參考：《青島大學(xué)》2016年博士論文

【摘要】：目的:海兔素(Aplysin)是從海洋三列凹頂藻中提取的脂溶性化合物,屬于溴代倍半萜。本實驗室研究證實海兔素具有多樣生物學(xué)活性,主要包括抗氧化、抗炎、抗腫瘤、免疫增強等。并且初步發(fā)現(xiàn)其對酒精誘導(dǎo)肝損傷大鼠具有保護效果,機制與調(diào)控線粒體介導(dǎo)的肝細胞凋亡有關(guān)。本課題在此研究基礎(chǔ)之上,從海兔素調(diào)控Kupffer細胞中TLR4信號轉(zhuǎn)導(dǎo)通路與調(diào)節(jié)腸道菌群兩個方面,繼續(xù)探討海兔素保肝功能與作用機制。方法:1.海兔素改善慢性酒精性肝損傷的效果評價(1)動物分組及模型建立:本實驗選取兩月齡健康雄性Wistar大鼠,共60只,體重大約180-220g,根據(jù)體重進行隨機數(shù)字表法分組,隨機分為3組,每組各20只。酒精模型組給予56度白酒(北京紅星二鍋頭)8 ml/(kg·bw·d),灌胃2周后,劑量提升到12 ml/(kg·bw·d),繼續(xù)灌胃6周;海兔素組酒精劑量同模型組,同時每日給予海兔素150 mg·kg-1·d-1灌胃,持續(xù)8周;正常組以等體積生理鹽水灌胃,持續(xù)8周。末次灌胃12小時后,使用代謝籠留取糞便,大鼠稱重后給予7%水合氯醛麻醉,腹主動脈取血,剝?nèi)「谓M織及小腸組織。(2)肝臟病理學(xué)檢查:HE染色觀察大鼠肝臟組織病理學(xué)改變及透射電鏡觀察大鼠肝細胞超微結(jié)構(gòu)變化。(3)肝功能評價:采用賴氏法檢測血清谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)活性;PNP比色法檢測血清堿性磷酸酶(ALP)活性;采用羥胺三氯化鐵比色法檢測血清膽堿酯酶(CHE)活性;采用IFCC速率法檢測血清谷氨酰轉(zhuǎn)移酶(GGT)活性。2.海兔素對慢性酒精性肝損傷大鼠Kupffer細胞吞噬能力和TLR4信號轉(zhuǎn)導(dǎo)通路影響(1)原代Kupffer細胞獲取:采用大鼠門靜脈膠原酶Ⅳ原位灌注法和Percoll密度梯度離心法分離大鼠肝臟Kupffer細胞,所得Kupffer細胞在37℃和5%CO2條件下進行培養(yǎng)。(2)ED2免疫化學(xué)染色鑒定Kupffer細胞純度,Giemsa染色觀察Kupffer細胞形態(tài),墨汁吞噬實驗檢測Kupffer細胞吞噬能力,MTT實驗檢測Kupffer細胞活力水平。(3)采用顯色鱟試劑檢測血漿內(nèi)毒素水平。(4)逆轉(zhuǎn)錄PCR法檢測Kupffer細胞中TLR4信號轉(zhuǎn)導(dǎo)通路關(guān)鍵蛋白CD14,TLR4和NF-κB m RNA水平的表達。(5)Western Blot法檢測CD14,TLR4蛋白表達水平。(6)ELISA法檢測Kupffer細胞中TLR4通路下游炎性細胞因子TNF-α和IL-1β含量。3.海兔素對慢性酒精性肝損傷大鼠腸道菌群的影響(1)小腸病理學(xué)檢查:HE染色觀察小腸上皮組織病理學(xué)改變,透射電鏡對大鼠小腸上皮細胞超微結(jié)構(gòu)進行觀察。(2)大鼠糞便腸道菌群基因組DNA提取:采用特異性結(jié)合DNA的離心吸附柱和抑制劑吸附片Inhibit EX提取糞便腸道菌群基因組DNA。(3)腸道菌群代表菌株定量分析:采用實時熒光定量PCR技術(shù)對糞便大腸桿菌、糞腸球菌、雙歧桿菌和乳酸桿菌16S r DNA V3可變區(qū)進行定量分析。(4)腸道菌群菌株數(shù)量與肝功酶學(xué)指標相關(guān)性分析:使用SPSS軟件,對腸道菌群的菌株數(shù)量與肝功酶學(xué)指標作Pearson相關(guān)性分析。結(jié)果:1.海兔素改善慢性酒精性肝損傷大鼠的效果評價HE染色結(jié)果顯示與正常對照組相比,酒精模型組中央靜脈出現(xiàn)大量炎性細胞浸潤,脂肪變性明顯且發(fā)現(xiàn)Mallory小體。正常對照組和海兔素干預(yù)組肝小葉結(jié)構(gòu)正常,肝細胞索以中央靜脈為中心呈現(xiàn)輻射狀,沒有發(fā)現(xiàn)典型脂肪變性和炎性細胞浸潤。透射電鏡觀察發(fā)現(xiàn)相比正常對照組,酒精模型組內(nèi)質(zhì)網(wǎng)擴張明顯,出現(xiàn)大量溶酶體,正常對照組與海兔素干預(yù)組核膜光滑,核孔清晰,線粒體結(jié)構(gòu)完整,但海兔素組出現(xiàn)少量脂滴。通過對血清中五種肝功能酶學(xué)指標檢測發(fā)現(xiàn),模型對照組血清中ALT,AST,ALP,CHE和GGT活性水平相對正常對照組分別顯著增高(P0.05);而海兔素干預(yù)組血清中ALT,AST,ALP,CHE和GGT活性水平相對酒精模型組分別顯著降低(P0.05)。2.海兔素對慢性酒精性肝損傷大鼠Kupffer細胞吞噬能力和TLR4信號轉(zhuǎn)導(dǎo)通路影響原代培養(yǎng)Kupffer細胞呈現(xiàn)典型梭子形狀或者不規(guī)則三角形形狀。ED2免疫化學(xué)染色發(fā)現(xiàn)Kupffer細胞純度達到97%以上。Giemsa染色結(jié)果發(fā)現(xiàn)Kupffer細胞表現(xiàn)出典型“荷包蛋”形狀。墨跡吞噬實驗發(fā)現(xiàn)各組Kupffer細胞胞漿中均可見吞噬的碳素顆粒,正常對照組細胞多維持細胞原有形狀,呈多角形、菱形等不規(guī)則形狀,酒精模型組細胞胞漿中可見大量碳素顆粒積聚,大部分細胞脹大呈圓形或橢圓形,Kupffer細胞吞噬能力明顯下降;海兔素干預(yù)組多數(shù)細胞維持原有形狀,部分細胞脹大呈圓形,吞噬能力相對酒精模型組明顯回升。MTT實驗結(jié)果顯示酒精模型組Kupffer細胞活力較正常對照組明顯增強,而海兔素組Kupffer細胞活力較酒精模型組顯著下降。酒精模型對照組血漿內(nèi)毒素水平相對正常對照組顯著升高20%(P0.05),海兔素組血漿內(nèi)毒素水平相對酒精模型組顯著下降7.1%(P0.05)。逆轉(zhuǎn)錄PCR結(jié)果顯示Kupffer細胞中酒精模型組CD14,TLR4和NF-κB m RNA表達相對正常對照組分別上升(1.1倍,1.7倍和2.0倍,P0.05)。海兔素組中CD14,TLR4和NF-κB m RNA表達水平相對酒精模型組顯著降低38%,20%和23%。Western Blot結(jié)果顯示,酒精模型組大鼠肝臟CD14,TLR4蛋白表達水平相較正常對照組顯著性上升1.25倍和1.13倍,海兔素干預(yù)組CD14,TLR4蛋白表達水平相較酒精模型組顯著性下降23%和20%。ELISA結(jié)果顯示酒精模型組TNF-α和IL-1β含量相較正常對照組分別顯著提高2.1倍和1.7倍,海兔素組TNF-α和IL-1β含量相較酒精模型組分別下降39%和31%(P0.05)。3.海兔素對慢性酒精性肝損傷大鼠腸道菌群的影響HE染色結(jié)果顯示酒精模型組中小腸絨毛結(jié)構(gòu)發(fā)生明顯萎縮。透射電鏡觀察到酒精模型組細胞連接變寬,微絨毛結(jié)構(gòu)稀疏,海兔素組出現(xiàn)大量溶酶體。菌株定量分析顯示相對正常對照組,酒精模型組大腸桿菌(P0.05)數(shù)量顯著升高,糞腸球菌數(shù)量呈上升趨勢(P0.05)。相對酒精模型組,海兔素組大腸桿菌數(shù)量顯著下降(P0.05),糞腸球菌數(shù)量呈下降趨勢(P0.05)。另外相對正常對照組,酒精模型組乳酸桿菌與雙歧桿菌數(shù)量都顯著下降(P0.05),海兔素干預(yù)后,乳酸桿菌數(shù)量呈上升趨勢(P0.05),雙歧桿菌數(shù)量顯著升高(P0.05)。通過4個代表菌株數(shù)量與肝功能酶學(xué)指標作相關(guān)性分析結(jié)果發(fā)現(xiàn)大腸桿菌數(shù)量與血清谷丙轉(zhuǎn)氨酶活性呈正相關(guān)的高度相關(guān)關(guān)系(P0.05)。結(jié)論:1.根據(jù)肝臟病理學(xué)觀察和肝功能酶活性檢測,海兔素對慢性酒精性肝損傷大鼠具有改善作用。2.海兔素通過增強慢性酒精性肝損傷大鼠肝臟Kupffer細胞吞噬能力,加大對內(nèi)毒素清除,抑制Kupffer細胞中TLR4信號轉(zhuǎn)導(dǎo)通路的激活,降低CD14,TLR4和NF-κB表達和下游炎性因子TNF-α和IL-1β含量達到保肝效果。3.海兔素對慢性酒精性肝損傷大鼠小腸同樣起保護效果;海兔素保肝效果可能與其調(diào)節(jié)腸道菌群有關(guān);大腸桿菌可能是海兔素調(diào)節(jié)腸道菌群改善慢性酒精性肝損傷大鼠的相關(guān)菌株。
[Abstract]:Objective: hahnin (Aplysin) is a fat soluble compound extracted from three marinas in the sea and belongs to brominated sesquiterpene. This laboratory study confirmed that hahnin has a variety of biological activities, mainly including antioxidant, anti-inflammatory, anti-tumor, immune enhancement and so on. And it has been found to have protective effect on alcohol induced liver injury in rats. It is related to the regulation of mitochondria mediated hepatocyte apoptosis. On the basis of this study, we continue to explore the function and mechanism of Rabbit Hare liver preservation from the two aspects of the TLR4 signal transduction pathway and the regulation of intestinal microflora in Kupffer cells. Methods: the evaluation of the effect of 1. hare on the improvement of slow alcoholic liver injury (1) animal score. Group and model establishment: a total of 2 month old healthy male Wistar rats were selected, with a total of 60 rats with a weight of about 180-220g. According to the weight, random numbers were divided into 3 groups and 20 were randomly divided into 3 groups. The alcohol model group was given 56 degree liquor (Beijing red Star Erguotou) 8 ml/ (kg. BW. D). After gavage for 2 weeks, the dose was promoted to 12 ml/ (kg. BW d) and continued. Continue After 6 weeks gavage, the alcohol dose in the hare group was the same as that in the model group. At the same time, the rabbits were given the hare 150 mg. Kg-1. D-1 for 8 weeks. The normal group was given the stomach with equal volume of normal saline for 8 weeks. After the last perfusion of the stomach for 12 hours, the excrement was taken by the metabolic cage, the rats were weighed with 7% chloral anaesthesia, the abdominal aorta was taken blood, and the liver tissue and small intestine were stripped. Organization. (2) liver pathological examination: HE staining observed the pathological changes of liver tissue and ultrastructural changes in rat liver. (3) liver function evaluation: serum glutamic prop aminotransferase (ALT) and cereal transaminase (AST) activity were detected by Ryan's method; PNP colorimetry was used to detect the activity of serum alkaline phosphatase (ALP); hydroxylamine three chloride was used. Serum cholinesterase (CHE) activity was detected by ferric colorimetry, and IFCC rate method was used to detect the effect of serum glutamyl transferase (GGT) activity.2. hare on the phagocytosis of Kupffer cells and TLR4 signal transduction pathway of Kupffer cells in rats with chronic alcoholic liver injury (1) primary Kupffer cells: using rat portal vein collagenase IV in situ perfusion and Percoll Kupffer cells of rat liver were separated by density gradient centrifugation. The obtained Kupffer cells were cultured at 37 and 5%CO2. (2) ED2 immunochemistry staining was used to identify the purity of Kupffer cells, Giemsa staining was used to observe the morphology of Kupffer cells, the phagocytic energy of Kupffer cells was detected by the ink phagocytosis test, and the MTT test was used to detect the activity level of Kupffer cells. (3) The level of plasma endotoxin was detected by chromogenic limulus reagent. (4) the expression of TLR4 signal transduction pathway key protein CD14, TLR4 and NF- kappa B m RNA level in Kupffer cells was detected by reverse transcriptase PCR. (6) Western Blot method was used to detect the expression level of CD14 and protein. The effect of hare on intestinal flora of rats with chronic alcoholic liver injury (1) pathological examination of small intestine: Observation of pathological changes of intestinal epithelium by HE staining, ultrastructure of small intestinal epithelial cells in rats by transmission electron microscopy. (2) extraction of genomic DNA from fecal intestinal flora of rats: centrifuge column and inhibition by specific combination of DNA Inhibit EX extraction of fecal intestinal microflora DNA. (3) intestinal microflora representative strains quantitative analysis: the quantitative analysis of fecal Escherichia coli, Enterococcus faecalis, bifidobacteria and Lactobacillus 16S R DNA V3 variable region by real-time fluorescence quantitative PCR. (4) the correlation analysis of the number of intestinal microflora and liver function enzymology index: SPSS software was used to analyze the Pearson correlation between the number of bacteria in the intestinal flora and the liver function enzymology index. Results: 1. the effect of hare on the improvement of chronic alcoholic liver injury in rats. The results of HE staining showed that there was a lot of inflammatory cell infiltration in the central vein of the alcohol model group compared with the normal control group, and the fatty degeneration was obvious and the Mallor was found. The hepatic lobules in the normal control group and the hare intervention group were normal, the hepatic cord was radiated with the central vein as the center, and no typical fatty degeneration and inflammatory cell infiltration were found. The transmission electron microscope observed that the endoplasmic reticulum of the alcohol model group showed a large number of lysosomes, the normal control group and the hare, compared with the normal control group. In the vegetarian intervention group, the nuclear membrane was smooth, the nuclear pore was clear and the structure of mitochondria was complete, but a small amount of lipid droplets appeared in the hare group. The serum levels of ALT, AST, ALP, CHE and GGT were found to be Zhuo Zenggao (P0.05) in the serum of the model control group compared with the normal control group, and the serum ALT, AST, ALP, C in the hare intervention group. The activity level of HE and GGT decreased significantly compared with the alcohol model group (P0.05) the phagocytosis of Kupffer cells and the TLR4 signal transduction pathway in the Kupffer cells of the rats with chronic alcoholic liver injury were influenced by the TLR4 signal transduction pathway, and the original culture Kupffer cells presented the typical shuttle shape or the irregular triangle shape.ED2 immunochemical staining found that the purity of Kupffer cells was reached. More than 97%.Giemsa staining results showed that Kupffer cells showed a typical "Lotus egg" shape. The phagocytic carbon particles were found in the cytoplasm of Kupffer cells in each group. The cells in the normal control group maintained the original shape of the cells, were polygonal, rhombus and other irregular shapes, and a large amount of carbon was found in the cytoplasm of the alcohol model group. The accumulation of prime particles, most of the cells swelled round or oval, the phagocytosis of Kupffer cells decreased obviously, most of the cells in the hare intervention group maintained the original shape, some cells swelled and round, and the phagocytic ability compared with the alcohol model group.MTT experimental results showed that the activity of Kupffer cells in the alcohol sperm model group was more obvious than that of the normal control group. The activity of Kupffer cells in the hare group was significantly lower than that in the alcohol model group. The plasma endotoxin level in the alcohol model control group was significantly increased by 20% (P0.05), and the plasma endotoxin level in the hare group decreased by 7.1% (P0.05) significantly (P0.05). The reverse transcriptase PCR results showed that the alcohol model group in Kupffer cells was CD14, TLR The expression of 4 and NF- kappa B m RNA increased (1.1 times, 1.7 times and 2 times, P0.05). The RNA expression level of CD14, TLR4 and NF- kappa B m in the hare group was significantly lower than that of the alcohol model group by 38%, 20% and 23%.Western results showed that the level of protein expression in the alcohol model group was significantly higher than that of the normal control group. 1.25 times and 1.13 times the CD14, the expression level of TLR4 protein in the hare intervention group was significantly lower than that of the alcohol model group by 23% and 20%.ELISA results showed that the content of TNF- A and IL-1 beta in the alcohol model group was significantly increased by 2.1 and 1.7 times compared with the normal control group. The TNF- alpha and IL-1 beta content in the hare group decreased by 39% and 31% (P0.05), respectively. The effect of.3. hare on the intestinal flora of rats with chronic alcoholic liver injury HE staining showed that the structure of small intestinal villi in the alcohol model group was obviously atrophied. The transmission electron microscope showed that the cell connections in the alcohol model group became wider, the microvilli structure was sparse, and the hare group appeared a large number of lysosomes. The number of Escherichia coli (P0.05) in the alcohol model group increased significantly and the number of Enterococcus faecalis increased (P0.05). Compared with the alcohol model group, the number of Escherichia coli in the hare group decreased significantly (P0.05) and the number of Enterococcus faecalis declined (P0.05). In addition, the number of lactobacillus and Bifidobacterium in the alcohol model group decreased significantly (P0.05). P0.05), the number of Lactobacillus increased significantly (P0.05) and the number of bifidobacteria increased significantly (P0.05). The correlation analysis between the number of 4 representative strains and the hepatic functional enzymology indicators found that the number of Escherichia coli was highly correlated with the activity of serum glutamic pyruvic aminotransferase (P0.05). Conclusion: 1. according to the liver Histopathological observation and detection of liver function enzyme activity, hahnin has an improved effect on chronic alcoholic liver injury in rats..2. hahnin can enhance the phagocytosis of Kupffer cells in liver of rats with chronic alcoholic liver injury, increase the clearance of endotoxin, inhibit the activation of TLR4 signal transduction pathway in Kupffer cells and reduce the expression of CD14, TLR4 and NF- kappa B The TNF- alpha and IL-1 beta content of downstream inflammatory factors can protect the liver from the effect of.3. hahnin on the small intestine of rats with chronic alcoholic liver injury, and the effect of hhnin protecting liver may be related to the regulation of intestinal flora, and E. coli may be the related strain of the rabbit rabbit with the regulation of intestinal microflora to improve the chronic alcoholic liver injury in rats.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R285.5

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